The most abundant species of RNAs found in cells are the ribosomal RNAs (rRNA). In animal cells, rRNAs are synthesized in specialized dense nucleolar bodies which are found in the nucleus of most eukaryotes. Transcription of 45S precursor rRNA is carried out by the nucleolar RNA polymerase I and the mature 18S and 28S ribosomal RNAs are produced after post-transcriptional processing of the primary transcripts. It is well established that the rate of rRNA transcription is acutely sensitive to changes in the environment and to the physiological state of the cell. Despite the importance of rRNA in maintaining the living state, little is known about the molecular basis underlying the regulation of rRNA transcription. Here, we propose to investigate the control of rRNA synthesis in human cells. We will use a cell-free RNA polymerase I transcription system derived from HeLa cells to study the regulation of human ribosomal RNAs synthesis. First, we will define the promoter region by mapping the control sequences using a novel site-directed mutagenesis procedure to generate insertion, deletion, and clustered point mutations. Next, we will attempt to isolate and identify specific transcription factors required to impart promoter selectivity to RNA polymerase I. This will be accomplished by biochemical fractionation of the cell-free transcription system with RNA polymerase I and specific transcription factors. We will also determine how these transcription factors operate by investigating their ability to bind specific promoter sequences and potential interactions with the RNA polymerase I subunits. Finally, we will investigate the mechanism governing changes in the rate of ribosomal RNA synthesis when cells respond to specific metabolic and physiologic changes brought about by viral infection.